P717: AG946, A PYRUVATE KINASE (PK) ACTIVATOR IMPROVES PK PROPERTIES AND RED BLOOD CELL (RBC) METABOLISM UPON EX VIVO TREATMENT OF RBCS FROM PATIENTS WITH MYELODYSPLASTIC SYNDROMES
Authors: Jonathan de Wilde, Titine Ruiter, Brigitte van Oirschot, Judith Jans, Lenny Dang, Megan Wind-Rotolo, Wouter van Solinge, Anna van Rhenen, Richard Van Wijk, Minke Rab
Journal: HemaSphere (2023)

Background: Myelodysplastic syndromes (MDS) consist of a heterogenous group of clonal myeloid malignancies, characterized by dysplasia, ineffective hematopoiesis and cytopenias. MDS patients frequently suffer from anemia. This directly affects quality of life and the limited therapeutic options often do not yield sufficient clinical improvement. Interestingly, decreased activity of red blood cell (RBC) pyruvate kinase (PK), a key regulatory enzyme of glycolysis, has previously been reported in patients with MDS. In light of current advances in the use of small molecule PK activators for treatment of RBC disorders we investigated the effect of ex vivo treatment of MDS RBCs with the PK activator AG946. Aims: To evaluate RBC PK and cellular properties of patients with MDS, and to determine the effect of ex vivo treatment with the PK activator AG946. Methods: Ten non-transfusion dependent MDS patients and six healthy controls (HCs) were studied. Baseline PK activity and PK thermostability were measured in purified RBCs and RBC lysates, respectively. Hexokinase (HK) activity was included to evaluate PK activity in relation to mean RBC age. Ex vivo treatment was performed by incubating MDS RBCs for 16 hours at 37 °C in presence or absence of the PK activator AG946 (10μM or 5μM) using dimethylsulfoxide (DMSO) as a blank. Effect of treatment was assessed by measuring PK activity and levels of adenosine triphosphate (ATP) (LC-MS/MS). Functional RBC analysis was performed by osmotic gradient ektacytometry (LoRRca MaxSis). To investigate the effect on PK thermostability RBC lysates were incubated with AG946 (2μM or 1μM, DMSO as blank). The effect of ex vivo treatment of AG946 on erythroid development was studied using peripheral blood mononuclear cells, cultured in MethoCultTM H4434 medium for 14 days in the presence or absence of AG946 (10μM, 625nM or 0.97nM, DMSO as blank). Results: Mean PK/HK ratio was significantly lower in RBCs from MDS patients compared to HCs (6.1 versus 10.5, p<0.001, Figure 1A). Upon ex vivo treatment, PK activity significantly increased in a non-dose-related manner compared to DMSO (10μM AG946, mean increase 29%; 5μM, 27%, Figure 1B). MDS RBCs displayed decreased PK thermostability at baseline (not shown), which could be restored by ex vivo treatment with AG946 (DMSO, 32% residual activity, 2μM AG946, 84%; 1μM, 86%, Figure 1C). Increase in activity was accompanied by significantly increased ATP levels (Figure 1D). On a functional level, RBC properties improved as reflected by the modest yet significant increase in Ohyper obtained by osmotic gradient ektacytometry, indicating improved hydration status (10μM AG946, mean increase 2.3%; 5μM, 2.8%). To date, culture assays were performed for 4/10 patients to investigate the effect of PK activation at the level of erythroid development; 1/4 showed a 36% increase (non-dose-related) in number of burst-forming units-erythroid (BFU-Es) upon treatment with AG946 (10μM, 625nM and 0.97nM) compared to DMSO. Summary/Conclusion: Our findings suggest that RBCs from MDS patients show a decrease in PK activity and thermostability. Furthermore, we show that ex vivo treatment with AG946 increases PK activity, ATP levels and stabilizes PK. Moreover, ex vivo treatment with AG946 improves RBC hydration, suggesting that improved energy status directly improves RBC functional properties. To better understand the effects on erythroid development additional investigation is required. In conclusion, our data warrant further investigation into the use of PK activator therapy as a potential novel therapeutic option for MDS. Image:Keywords: Red blood cell, Pyruvate kinase, Myelodysplastic syndrome